The centromere, a highly specialized region found on each chromosome, plays a critical role in the segregation of chromosomes. Failure to maintain proper centromeric function results in aneuploidy, leading to various diseases, such as cancer. However, the centromeres represent the least characterized region of eukaryotic genomes. All eukaryotic centromeres studied to date are packaged into a specialized chromatin domain that contains centromeric histone H3 (cenH3). The long-term goal of this proposal is to understand how proper centromeric structure is formed. Centromeric chromatin is arranged in interspersed blocks of cenH3 and canonical H3 nucleosomes in flies and humans, which is hypothesized to aid in three-dimensional folding of the centromeric region; however, the regulation of DNA replication and assembly of centromeric chromatin are poorly understood. In this proposal, 1) physical relationships between cenH3 and DNA replication / chromatin assembly factors will be investigated in Drosophila; and 2) cenH3 associated sequence will be isolated. Characterization of both protein factors and DNA elements that act in concert at the centromeres will lead to better understanding of centromere formation. [unreadable] [unreadable]